1. Volume 85, Issue 4, Pages 855-870 (April 2014)
Klotho has dual protective effects on cisplatin-induced acute kidney injury 
Monica C. Panesso, Mingjun Shi, Han J. Cho, Jean Paek, Jianfeng Ye, Orson W. Moe, Ming Chang Hu 
Kidney International 
Volume 85, Issue 4, Pages (April 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Cisplatin (CP) induces acute kidney injury. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (10mg/kg body weight) or vehicle (same volume of 0.9% NaCl) once into mice with three different Klotho genetic backgrounds. (a) Plasma creatinine (PCr) and (b) blood urea nitrogen (BUN) were measured at days 0, 2, 4, 7, and 14 post injection. The results are expressed as means±s.d. of eight animals from each group. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05, **P<0.01 versus WT cisplatin injection and #P<0.05, ##P<0.01 versus Tg-Kl cisplatin injection. At days 4, 7, and 14 post cisplatin or vehicle injections, mice were euthanized, and the kidneys were harvested and sectioned for histology from four animals in each group. (c) Representative hematoxylin and eosin (H&E) staining in paraffin-embedded kidney sections. Renal tubular casts (asterisk); dilated renal tubules (arrow head); and infiltrated inflammatory (arrow). (d) Kidney histological scores were obtained from H&E staining of the kidneys by a nephropathologist blinded to the experimental conditions. Results of pathological scores are expressed as means±s.d. of eight animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 and **P<0.01 between the two groups. Kl/+, Klotho hypomorphic; Tg-Kl, transgenic Klotho overexpressing; WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Cisplatin induces Klotho deficiency and increases neutrophil gelatinase-associated lipocalin (NGAL) expression. (a) Representative immunoblot for NGAL and Klotho in total kidney lysate in each group (total four mice per group) at days 4, 7, and 14 post cisplatin (CP) or vehicle injection. (b) Summarized densitometric analyses of all samples from vehicle or cisplatin-injected mice. Data are expressed as means±s.d. of four animals from each group. (c) Representative fluorescent immunohistochemistry for Klotho (blue) and NGAL (green) in paraffin kidney sections at days 4, 7, and 14 post injection (four mice in each group). Arrows show NGAL signals. (d) Levels of Klotho and NGAL transcripts in the kidneys from vehicle or cisplatin-injected mice on the fourth, seventh, and fourteenth day were analyzed by quantitative PCR. The relative quantity of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin vehicle-injected wild-type (WT) mice as reference in each time point. Data are expressed as means±s.d. of six animals from each group. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 and **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis. (a) Representative fluorescent immunohistochemistry for TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; a cell apoptosis marker, green signal) and DAPI (4,6-diamidino-2-phenylindole; a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (four mice per group) at days 4, 7, and 14 post-cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (b) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at × 40 magnification. Data are expressed as means±s.d. of four animals from each group. (c) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from four mice kidneys in each group at day 7 post-cisplatin injection. (d) Summarized densitometric analysis of all samples from vehicle or cisplatin-injected mice. Data are expressed as means±s.d. of four animals from each group. (e) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin-injected mice on the seventh day were analyzed by quantitative PCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared with vehicle-injected wild-type (WT) mice in each study time point. Data are expressed as means±s.d. of six animals from each group. (f) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (four mice per group) at days 0, 4, 7, and 14 post-cisplatin or vehicle injection. Arrows show positive signal of Ki67. (g) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at × 40 magnification. Data are expressed as means±s.d. of four animals from each group. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 and **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Klotho protects normal rat kidney (NRK) cells from cisplatin cytotoxicity. NRK cells were plated on Transwell plates to allow separate access to apical and basal compartments. Cisplatin (CP), Klotho (Kl), or cimetidine (Cim) were added to either apical or basolateral compartments simultaneously. Alternatively, cisplatin was added basally for 20min followed by wash-out and addition of cimetidine or Klotho to basal side. (a) Cell damage was measured by lactate dehydrogenase (LDH) release. (b) Representative fluorescent immunocytochemistry for TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling; apoptosis marker, green) and DAPI (4,6-diamidino-2-phenylindole; marker of cell nuclei, blue) in NRK cells from a total of four independent experiments. Arrows show positive TUNEL signal. (c) Summary of positive TUNEL cells divided by the total number of DAPI cells from five microscopic fields at × 40 magnification. In both (a) and (c), data are expressed as means±s.d. of four independent experiments. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 and **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Klotho (Kl) suppresses organic cation transporter (OCT)-mediated cisplatin (CP) update by normal rat kidney (NRK) cells. (a) Representative immunoblots for OCT1 and OCT2 protein expression in total cell lysate of NRK cells from three independent experiments. (b) Representative reverse transcriptase–PCR for oct1, oct2, and β-actin transcripts in NRK cells and kidneys of normal Sprague-Dawley rat. Same findings were seen in three independent experiments. (c) Representative confocal images showing fluorescence-labeled cisplatin uptake by NRK cells. Cisplatin was labeled with Oregon Green 488 dye (Invitrogen) and added to apical or basolateral medium with Klotho or cimetidine (Cim) or vehicle. Arrows show the presence of cisplatin in the cytoplasmic compartment of NRK cells. Same results were seen in three independent experiments. (d) 14C-tetraethylammonium (14C-TEA) was basally or apically incubated with cimetidine or Klotho simultaneously or 14C-TEA was basally incubated for 20min followed by wash-out and addition of cimetidine or Klotho to the basal medium. Data are expressed as means±s.d. of four independent experiments. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Mechanism of action of Klotho on OCT2 in Chinese hamster ovary (CHO) cells. CHO cells were transiently transfected with empty pcDNA3.1 vector, human OCT2-V5 plasmid, or human Klotho plasmid. Two days post transfection, cells were subjected to 14C-tetraethylammonium (14C-TEA) uptake, immunocytochemistry, immunoblot, and quantitative reverse transcriptase–PCR (RT-qPCR). β-Glucuronidase (β-glu; 100μg/ml), sialidase (0.1IU/ml), DSAL (D-Saccharic acid 1,4-lactone; 10μmol/l), or cimetidine (0.1mmol/l) were added into culture medium 16h before assays. (a) 14C-TEA uptake by CHO cells. Data are expressed as means±s.d. of four independent experiments. (b) Representative image (X, Y, and Z scanning) of fluorescent immunocytochemistry for V5 (OCT2, green), Klotho (blue), and phalloidin (marker of actin, red) in CHO cells from four independent experiments. CHO cells without Klotho expression had strong OCT2 expression (shown by arrowhead), whereas CHO cells expressing high Klotho had weak OCT2 expression (shown by arrow). (c) Summary of arbitrary units of OCT2 signal versus Klotho signal in OCT2/V5 and/or in Klotho transiently transfected CHO cells. Each point was an average of arbitrary units from five different randomized fields where each field has at least four positive transfected cells at × 100 magnification. OCT2-positive cells (green symbols); Klotho-positive cells (blue symbols); and double positive cells (yellow symbols). Y axis is an arbitrary unit of OCT2 signal calculated by green density (OCT2) over red density (actin) using the Image J program; X axis is an arbitrary unit of Klotho signal, which was calculated by green density (OCT2) over red density (actin) using the Image J program. Data are expressed as means±s.d. of four independent experiments. (d) Representative immunoblots for OCT2/V5 by V5 antibody, Klotho by KM2076 antibody, and β-actin in total membrane protein extracted from CHO cells. Identical results were seen in three independent experiments. Bottom panel is a summary of densitometric analysis of bands of V5 and β-actin from all three independent experiments. (e) Levels of human OCT2, rat oct1, and oct2 mRNA in NRK cells transiently transfected with human OCT2/V5 plasmid and Klotho plasmid were quantitatively analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared with OCT2/V5–transfected CHO cells. Data are expressed as means±s.d. of three independent experiments. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 or **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Klotho decreased cationic styryl dye (ASP+, a substrate of organic cation transporter) uptake by kidney slices. Kidney slices from Kl/+, wild-type (WT), and Tg-Kl mice were co-incubated with ASP+ with vehicle, Klotho, or cimetidine for 2h. Kidney sections were photographed for ASP+ signal (green fluorescent) with laser confocal microscopy.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

9. Figure 8
OCT1 and OCT2 are expressed in mouse kidney. (a) Representative fluorescent immunohistochemistry for OCT1 and OCT2 (green), Klotho (blue), and phalloidin (actin, red) in paraffin-embedded kidney sections from wild-type (WT) mice. (b) Representative immunoblots for OCT1, OCT2, and β-actin in total kidney lysate from three mice in each genotype. Right panel is a summarized densitometric analysis of OCT1 and OCT2 proteins over β-actin. Data are expressed as means±s.d. of three animals from each genotype. (c) Representative fluorescent immunohistochemical stain for OCT2 (green) and Klotho (blue) in paraffin-embedded kidney sections from mice in each genotype. (d) Representative reverse transcriptase–PCR (RT-PCR) for oct1, oct2, and β-actin transcripts in the kidney from mice with each genotype. (e) The levels of oct2 mRNA in mouse kidneys were quantitatively analyzed by RT-qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin compared with WT mice. Data are expressed as means±s.d. of three animals from each genotype. All experiments were repeated independently three times. Statistical significance was assessed by one-way analysis of variance followed by Student–Newman–Keuls test, and significant differences were accepted when *P<0.05 and **P<0.01 between the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

10. Figure 9
Klotho suppressed cisplatin excretion in vivo. Ten microliter per gram body weight of 14C-tetraethylammonium (14C-TEA; 0.1μCi/μl, 3.5mCi/mmol) was once injected intravenously into Kl/+, wild-type (WT), and Tg-Kl mice; blood and urine samples were collected at the indicated times. (a) Time course of blood concentration of 14C-TEA. (b) Time course of urinary excretion of 14C-TEA. (c) Cumulated urinary 14C-TEA over 60min. (d) Creatinine clearance. In panels (a–c), data are expressed as means±s.d. of three animals from each genotype, and statistical analysis were run by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test. Significant differences were accepted when *P<0.05, **P<0.01 compared with Kl/+ mice and #P<0.05, ##P<0.01 compared with WT mice. (e) Two hours after injection, eight organs were harvested and homogenized for measurement of 14C-TEA uptake and total protein. Uptake of 14C-TEA normalized to protein was calculated and significances between mice with three different genotypes were analyzed by one-way ANOVA followed by Student–Newman–Keuls test. Significant differences were accepted when *P<0.05 and **P<0.01 between the two groups. (f) Tissues and organs were sectioned at 10-μm thickness and subjected to autoradiography.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

11. Figure 10
Summary of findings and proposed scheme for Klotho effect on cisplatin-induced cytotoxicity. Klotho OCT2-independent effect is related to anti-apoptosis (right panel). Klotho OCT2-dependent effects (left panel) can be glucuronidase dependent or glucuronidase independent. Glucuronidase-independent effect is associated with downregulation of OCT2 mRNA. Glucuronidase-dependent effects are more complex. Klotho could decrease total cell OCT2 protein by functioning as a glucuronidase independent of OCT2 mRNA. Klotho also reduces primarily glycosylated surface OCT2 protein in 2h effect and both glycosylated and unglycosylated surface OCT2 in 2 days effect. Klotho can potentially directly decrease OCT2 activity without alteration of OCT2 protein (dashed line). Collectively, Klotho suppresses OCT2 transport function and consequently reduces cisplatin uptake. On the other hand, Klotho functions as an anti-apoptotic agent and as an anti-oxidant independently of cisplatin uptake to protect cells against cisplatin-induced cytotoxicity.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions